The lambda (lambda) replicator represents an essential cis-acting region of the DNA template. The activation and recognition of this region by replication proteins results in the initiation of lambda DNA synthesis. How many DNA target sites comprise the replicator? If there is more than one site does this mean there are multiple essential sites per replicator, or that there is more than one replicator site? I have isolated 51 new replicator mutations that prevent the initiation of lambda replication. The genetic mapping of these mutations suggest that they occur in at least four different locations. We have proposed to determine the precise location and physical characteristics of representative replicator mutations from each of these regions by DNA sequence analysis using the dideoxy technique. The replicator fragments will be isolated by first cloning in pBR322 and then into M13mp7. The activity of the replicator mutations will be determined by two additional experiments designed to test the effect of the replicator mutations (a) upon replication from alternative target sites and (b) on the synthesis of specific lambda transcripts whose positive regulation is coupled to the activation event for initiation of lambda replication.